Previous work relating to chemical carcinogenesis has demonstrated that carcinogenic potency of a compound often correlates with its mutagenic power. See McCann, J., Choi, E., Yamasaki, E. and Ames, B.N. Proc. Natl. Acad. Sci. USA 72: 5135-5139 (1975); McCann, J. and Ames, B.N. Proc. Natl. Acad. Sci.-USA 73: 950-954 (1976); Bridges, B.A. Nature 261 195-200 (1976); and, Bouck, N. and diMayorca, G. Nature 264: 722-727 (1976). This suggests that DNA is the ultimate target of carcinogenic activation. Because of this, researchers have attempted to identify and study DNA segments in tumor cells, often referred to as "oncogenes," whose alteration is critically important for oncogenic conversion.
One recent approach to isolation of an oncogene involved the transfer of tumor cell DNA from the EJ bladder carcinoma cell line into non-transformed NIH3T3 mouse fibroblasts. It was discovered that the phenotype of cellular transformation could be passed from cell to cell in this manner. Tumor DNA was able to induce foci of transformed cells in the recipient NIH monolayer culture while DNA from normal, untransformed donor cells failed to produce foci. See Shih, C., Shilo, B., Goldfarb, M.P., Dannenberg, A. and Weinberg, R.A. Proc. Natl. Acad. Sci. USA 76: 5714-5718 (1979); Cooper, G.M., Okenquist, S. and Silverman, L. Nature 284: 418-421 (1980); Shih, C., Padhy, L.C., Murray, M.J. and Weinberg, R.A. Nature 290: 261-264 (1981); Krontiris, T.G. and Copper, G.M. Proc. Natl. Acad. Sci. USA 78: 1181-1184 (1981); and, Perucho, M. et al. Cell 27: 467-476 (1981). These results demonstrated oncogenic factors present in the EJ tumor cell line DNA which were apparently absent from the DNA of normal cells.
Studies which examined the sensitivity or resistance of oncogenic DNA from the EJ bladder carcinoma line to treatment of various site-specific endonucleases indicated that certain specific donor DNA sequences were involved in such cellular transformation. See Lane, M.A., Sainten, A. and Cooper, G.M. Proc. Natl. Acad. Sci. USA 78: 5185-5189 (1981); and, Shilo, B. and Weinberg, R.A. Nature 289: 607-609 (1981). This concept of a discrete, definable oncogene was later directly demonstrated by molecular isolation of discrete transforming genes from the EJ human bladder carcinoma cell line by a method described in co-pending application Ser. No 379,721, filed May 19, 1982, now abandoned, in the name of Robert A. Weinberg.
As described in this co-pending application, DNA isolated from the EJ cell line was serially passed by transfection into NLH3T3 mouse fibroblast cells until a mouse fibroblast cell was selected containing essentially only the human bladder cancer oncogene and a marker. The marker used in this work was an Alu DNA sequence, which is repeated about 300,000 times in human DNA, but is not present in mouse fibroblast DNA. The interspecies transfection thus resulted in the ultimate selection of a cell containing the oncogene of interest and its associated marker. All DNA from this transfected cell was employed in the creation of a genomic library in a lambdaphage and the appropriate chimeric lambdaphage was then selected using a probe specific for the human Alu marker.
This work resulted in localization of the oncogenic activity for the EJ bladder carcinoma DNA to a 6.6 kb long DNA segment generated by the endonuclease BamHI. The 6.6 kb segment was cloned in the plasmid vector pBR322 and then used as a sequence probe in a southern blot analysis. This indicated that the oncogene derived from a sequence of similar structure present in the normal human genome. See Goldfarb, M., Shimizu, Perucho, M. and Wigler, M. Nature 296: 404-409 (1982); and, Shih, C. and Weinberg, R., Cell 29, 161-169 (1982). Thus, it appeared that the human bladder oncogene had arisen by mutation of a normal cellular gene during the process of carcinogenesis.
Comparison of the EJ bladder oncogene with its corresponding normal cellular sequence (the "proto-oncogene") was aided by the subsequent discovery that this oncogene was homologous to the transforming gene of the rat-derived Harvey murine sarcoma virus. See Der, C., Krontiris, T.G. and Cooper, G.M. Proc. Natl. Acad. Sci. USA 79: 3637-3640 (1982); Parada, L.F., Tabin, C.J., Shih, C. and Weinberg, R.A. Nature 297: 474-479 (1982); and, Santos, E. et al. Nature 298: 343-347 (1982). This rat sarcoma virus gene, termed v-Ha-ras, had been acquired from the rat genome during the process of formation of the chimeric viral genome. See Scolnick, E.M. and Parks, W.P. J. Virol. 13: 1211-1219 (1974); and, Shih, T.Y., Williams, D.R., Weeks, M.O., Maryak, J.M., Vass, W.C. and Scolnick, E.M. J. Virol. 27: 45-55 (1978). Both the rat and human cellular homologues of the v-Ha-ras have been isolated in the course of studies of this gene. See DeFeo, D., Gonda, M.A., Young, H.A., Change, E.H., Lowy, D.R., Scolnick, E.M. and Ellis, R.W. Proc. Natl. Acad. Sci. USA 78: 3328-3332 (1981); and, Chang, E.H., Gonda, M.A., Ellis R.W., Scolnick, E.M. and Lowy, D.R. Proc. Natl. Acad. Sci. USA, 79, 4848-4852 (1982).The human cellular homologue of the v-Ha-ras was found to correspond precisely to the normal antecedent of the EJ bladder oncogene. See Parada, L.F., Tabin, C.J., Shih, C. and Weinberg, R.A. Nature 297: 474-479 (1982).
Preliminary comparisons between the EJ oncogene and its normal cellular counterpart, termed c-Ha-ras, were made. See Ellis, R.W., DeFeo, D., Maryak, J.M., Young, H.A., Shih, T.Y., Chang, E.H., Lowy, D.R. and Scolnick, E.M. J. Virol. 36: 408-420 (1980). In this work, it was shown that a molecular clone of the normal cellular gene did not induce foci when applied to NLH3T3 monolayers, while a clone of the bladder oncogene exhibited a biological activity of ca. 5 .times.10.sup.4 foci forming units per microgram of transfected DNA. See Shih, C. and Weinberg, R.A. Cell 29: 161-169 (1982); and, Chang, E.H., Gonda, M.A., Ellis, R.W., Scolnick, E.M. and Lowy, D.R. Proc. Natl Acad. Sci. USA 79, 4848-52 (1982).
This stark difference in function did not correlate with any apparent structural differences between the two clones. Rough restriction endonuclease site mapping of the EJ oncogene clone and the uncloned related human proto-oncogene indicated that the two were basically indistinguishable over the 6.6 kb sequence which contained the transforming activity of the EJ oncogene. See Shih, C. and Weinberg, R.A. Cell 29: 161-169 (1982). Finer mapping was later made possible by the direct comparison of molecular clones of the two genes, but again, no differences using a series of different endonucleases were found, except for a single difference 3' (downstream) of the coding regions of the gene. This difference was interpreted to represent a functionally silent polymorphism of the gene present in the gene pool, of the type previous documented by others. See Goldfarb, M., Shimizu, Perucho, M. and Wigler, M. Nature 296: 404-409 (1982).
Thus, a drastic functional difference had been demonstrated for two structurally similar genes, the human bladder cancer oncogene and its normal proto-oncogene. No differences in the structure of the two genes was known, but it was theorized that there might be differences which produced functional differences in one of two ways. The alterations could involve a change in sequences regulating the expression of the gene, or alternatively, the transformed phenotype could be due to changes in the protein-encoding portion of the gene. The first hypothesis would likely produce up-regulation of transcription or translation of the gene, yielding high levels of an otherwise normal protein product, while the second hypothesis would suggest synthesis of an altered protein. Both types of alteration could also act in concert to create the observed difference in function.